WO1999061657A1 - Ensemble de reactifs liquides pour une determination quantitative du l-lactate - Google Patents

Ensemble de reactifs liquides pour une determination quantitative du l-lactate Download PDF

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Publication number
WO1999061657A1
WO1999061657A1 PCT/US1999/012174 US9912174W WO9961657A1 WO 1999061657 A1 WO1999061657 A1 WO 1999061657A1 US 9912174 W US9912174 W US 9912174W WO 9961657 A1 WO9961657 A1 WO 9961657A1
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Prior art keywords
reagent set
hydrogen
lactate
set according
agent
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PCT/US1999/012174
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English (en)
Inventor
Allan R. Dorn
Catherine J. Hurt
Larry D. Mountain
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Roche Diagnostics Corporation
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Priority to CA002334276A priority Critical patent/CA2334276C/fr
Priority to EP99953363A priority patent/EP1082453B1/fr
Priority to DE69940155T priority patent/DE69940155D1/de
Publication of WO1999061657A1 publication Critical patent/WO1999061657A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06F—ELECTRIC DIGITAL DATA PROCESSING
    • G06F2207/00—Indexing scheme relating to methods or arrangements for processing data by operating upon the order or content of the data handled
    • G06F2207/72—Indexing scheme relating to groups G06F7/72 - G06F7/729
    • G06F2207/7219—Countermeasures against side channel or fault attacks
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/962—Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/963—Methods of stopping an enzyme reaction or stabilizing the test materials
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/975—Kit

Definitions

  • the present invention is directed to sets of liquid reagents that provide calibration stability in an enzyme based spectrophotometric assay for the measurement of lactate in patient samples.
  • the present invention is also directed to a method for using the liquid reagents.
  • the chemical marker is a specific compound that is expressed in either abnormally high or low amounts during the course of an illness.
  • a clinician examines a patient's plasma and/or cerebrospinal fluid using an assay specifically designed to quantitatively determine the concentration of the marker.
  • a physician observing unusual concentrations of the marker understands that the patient may be suffering from an associated illness.
  • L-lactate is a chemical marker that is associated with a number of pathological states which result from a reduced oxygenation of biological fluids. Shock, pneumonia and congestive heart failure produce increased levels of L-lactate in plasma. One observes abnormally high cerebrospinal fluid levels of L-lactate as the result of bacterial meningitis, hypocapnia and cerebral ischemia.
  • L-lactate is oxidized to pyruvate by lactate oxidase (LOD).
  • LOD lactate oxidase
  • POD peroxidase
  • the concentration of the chromogen is measured spectrophotometrically. Because the concentration of chromogen is directly proportional to the concentration of lactate in the initial solution, one can calculate an observed lactate concentration.
  • a technician takes to run an enzyme based, spectrophotometric assay for L-lactate There are several steps that a technician takes to run an enzyme based, spectrophotometric assay for L-lactate. First a proper wavelength for the spectrophotometric measurement of the chromogen is chosen. Second, a "calibration factor" is established that allows the mathematical conversion of the spectrophotometric measurement of the chromogen to an observed L-lactate concentration. Plasma or cerebrospinal fluid sample from a patient is collected and prepared for testing. Finally, the prepared sample is mixed with the proper enzymatic reagents and there is a spectrophotometric measurement of the absorbence of radiation by the chromogen.
  • the technician establishes the calibration factor for L-lactate concentration by correlating the original concentration of L-lactate in a sample with the amount of radiation absorbed by the resulting chromogen. To establish this correlation, one has to be able to separate out the absorbence of the chromogen from the absorbence of other components in the assay system. This process can be represented by Scheme 2.
  • the technician combines known concentrations of the assay reagents: lactate oxidase, peroxidase, a hydrogen donor, a coupling agent, and any other chemical element needed to perform the spectrophotometric determination.
  • An aliquot is removed from the reagent combination and absorbence is read on a spectrophotometer.
  • the technician adds a predetermined amount of L-lactate and allows the enzymatic reactions providing the chromogen to proceed until they are essentially complete.
  • An aliquot of the chromogen containing solution is then removed and a second absorbence reading on the spectrophotometer is taken. The difference between the technician's final and initial spectrophotometric readings is the "calibration factor.”
  • a technician can employ a single set of reagents over an extended period of time; 90 days is not unusual where the technician refrigerates the open containers. Because the reagents are not stable, the calibration factor must be determined frequently during this time period. If one could calculate the calibration factor for a reagent set once, and then use that number for every spectrophotometric assay run with the reagent set, a substantial amount of time in performing an L-lactate assay would be saved. This time savings would allow a physician to more quickly and efficiently interpret the assay results and render a diagnosis.
  • a set of assay reagents can be either in a solid phase cartridge form or in a liquid form.
  • Solid phase cartridges are limited to use in specialized instruments designed to receive the cartridges.
  • Liquid reagents can be used in a variety of generalized instruments. This versatility allows hospital laboratories to maintain fewer instruments and therefore reduces the costs associated with laboratory testing.
  • a reagent is in liquid form, the user does not need to reconstitute it prior to performing the assay.
  • a technician can more quickly determine the L-lactate concentration in a biological fluid and also eliminate mixing errors associated with the reagent reconstitution.
  • the development of a liquid reagent set for an L-lactate assay that exhibits a relatively constant calibration factor therefore, meets an important need. It provides for a more efficient diagnosis of a set of critical medical conditions.
  • the present invention provides a liquid reagent set for an L-lactate spectrophotometric assay.
  • the liquid reagent set includes a lactate oxidase, a peroxidase, a hydrogen donor of structure 1, an agent that substantially prevents
  • the substituents on hydrogen donor 1 are defined as follows: Ri is hydrogen or alkyl; R 2 and R 3 are, independently, hydrogen, alkyl or aryl; R 4 is SO 3 M or CO 2 M, wherein M is hydrogen or a cation; and R 5 is hydrogen or alkyl.
  • the reagent set exhibits a calibration factor that varies by less than about 10% over a period of about 20 days, when the reagent set is stored at 4 °C.
  • the lactate oxidase is isolated from a microorganism.
  • the peroxidase is isolated from horseradish.
  • the substituents of the hydrogen donor are defined as follows: R] is alkyl; R 2 and R 3 are hydrogen; R 4 is SO 3 M, wherein M is a cation; and R 5 is alkyl.
  • the agent that substantially prevents ascorbic acid interference is a biological oxidizing agent.
  • the agent that substantially prevents bilirubin interference is a chemical oxidizing agent.
  • the coupling agent is 4-aminoantipyrine or a 4- aminoantipyrine analogue.
  • the buffer is a phosphate buffer.
  • the reagent set exhibits a calibration factor that varies by less than about 7% over a period of about 40 days.
  • the reagent set further comprises a preservative.
  • the reagent set exhibits a calibration factor that varies by less than about 7% over a period of about 40 days, and the substituents of the hydrogen donor are defined as follows: Ri is methyl; R and R are hydrogen; R 4 is SO 3 M, wherein M is sodium; and R 5 is methyl.
  • the reagent set further comprises a preservative, and the preservative is sodium azide.
  • the reagent set further comprises a preservative, and the preservative is sodium azide.
  • the substituents of the hydrogen donor are defined as follows: Rj is methyl; R and R 3 are hydrogen; R 4 is SO 3 M, wherein M is sodium; and R 5 is methyl.
  • the reagent set further comprises a preservative, and the preservative is sodium azide.
  • the buffer is a phosphate buffer, and the substituents of the hydrogen donor are defined as follows: Ri is methyl; R 2 and R 3 are hydrogen; R 4 is SO 3 M, wherein M is sodium; and R 5 is methyl.
  • the reagent set further comprises a preservative, and the preservative is sodium azide.
  • the buffer is a phosphate buffer, and the substituents of the hydrogen donor are defined as follows: Ri is methyl; R 2 and R 3 are hydrogen; t is SO M, wherein M is sodium; and R 5 is methyl.
  • This reagent set exhibits a calibration factor that varies by less than about 7% over a period of about 40 days.
  • the reagent set further comprises a preservative, and the preservative is sodium azide.
  • the buffer is a phosphate buffer, and the substituents of the hydrogen donor are defined as follows: Ri is methyl; R 2 and R 3 are hydrogen; R t is SO 3 M, wherein M is sodium; and R 5 is methyl.
  • This reagent set exhibits a calibration factor that varies by less than about 5% over a period of about 60 days.
  • the present invention also provides a method for determining the concentration of L-lactate in a biological fluid.
  • the method includes the following steps: collecting a biological fluid from a subject; and, performing a spectrophotometric assay on the biological fluid using a liquid reagent set.
  • the liquid reagent set in this embodiment includes a lactate oxidase, a peroxidase, a hydrogen donor of structure 1 , an agent that substantially prevents ascorbic acid interference, an
  • the substituents on hydrogen donor 1 are defined as follows: R ⁇ is hydrogen or alkyl; R and R 3 are, independently, hydrogen, alkyl or aryl; i is SO 3 M or CO 2 M, wherein M is hydrogen or a cation; and R 5 is hydrogen or alkyl.
  • the reagent set exhibits a calibration factor that varies by less than about 10% over a period of about 20 days, when the reagent set is stored at 4 °C.
  • the substituents of the hydrogen donor of the liquid reagent set used in the L-lactate determination method are defined as follows: Ri is methyl; R 2 and R 3 are hydrogen; t is SO 3 M, wherein M is a sodium ion; and R 5 is methyl.
  • Ri is methyl
  • R 2 and R 3 are hydrogen
  • t is SO 3 M, wherein M is a sodium ion
  • R 5 is methyl.
  • the present invention also provides a method for determining the concentration of
  • L-lactate in a biological fluid wherein a reagent set used in the determination includes a preservative.
  • the method includes the following steps: collecting a biological fluid from a subject; and, performing a spectrophotometric assay on the biological fluid using a liquid reagent set.
  • the liquid reagent set in this embodiment includes a lactate oxidase, a peroxidase, a hydrogen donor of structure 1 , an agent that substantially prevents ascorbic acid interference, an
  • the substituents on hydrogen donor 1 are defined as follows: Ri is hydrogen or alkyl; R 2 and R 3 are, independently, hydrogen, alkyl or aryl; R 4 is SO 3 M or CO 2 M, wherein M is hydrogen or a cation; and R 5 is hydrogen or alkyl.
  • the reagent set exhibits a calibration factor that varies by less than about 10% over a period of about 20 days, when the reagent set is stored at 4 °C.
  • the substituents of the hydrogen donor of the liquid reagent set used in the L-lactate determination method are defined as follows: Rj is methyl; R 2 and R are hydrogen; Ri is SO 3 M, wherein M is a sodium ion; and R 5 is methyl.
  • FIG. 1 shows a general structure for a hydrogen donor of the present invention.
  • Bio fluid refers to any liquid substance removed from a living organism.
  • the biological fluid will be either plasma or cerebrospinal fluid. More preferably, the biological fluid will be either plasma or cerebrospinal fluid from a human.
  • “Calibration factor” is a number that allows one to mathematically convert the spectrophotometric measurement of a chromogen to an observed L-lactate concentration. One obtains this number by correlating a known concentration of L-lactate in a sample with the amount of UV radiation absorbed by the chromogen which is produced from the reaction of L-lactate. This process is illustrated in Scheme 2.
  • “Hydrogen donor” refers to a compound that is oxidized in a chemical reaction, resulting in the formation of a chromogen in a spectrophotometric assay.
  • Hydrogen donors are known within the art, and examples of hydrogen donors include, but are not limited to, the following compounds: phenol, 4-chlorophenol, 2,4-dichlorophenol-6-sulphonic acid, N,N-dialkylaniline, N,N-dialkyl-m-toluidine, sodium salt of N-ethyl-N- sulphopropylaniline, sodium salt of N-ethyl-N-sulphopropyl-m-toluidine, sodium salt of N- ethyl-N-sulphopropyl-m-anisidine, sodium salt of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m- toluidine, sodium salt of N-ethyl-N-(2-hydroxy-3-sulfopropy)-m-anisidine, sodium salt of 3,5-dimethyl-N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline and sodium salt of 3,5-di
  • Coupling agent refers to a compound that couples to an oxidized hydrogen donor, resulting in the formation of a chromogen in a spectrophotometric assay.
  • Coupling agents are known within the art, and examples of coupling agents include, but are not limited to, the following compounds: 4-aminoantipyrine; trisubstituted 4-amino-3-pyrazoline-5-ones, such as l-(2,4,6-trichlorophenyl)-2,3-dimethyl-4-amino-pyrazoline-5-one and l-(3,5- dichlorophenyl)-2,3-dimethyl-4-amino-3-pyrazoline-5-one; and, 4-aminoantipyrine analogues, such as l-phenyl-2,3-dimethyl-4-dimethylamino-3-pyrazoline-5-one.
  • the coupling agent is 4-aminoantipyrine or a 4-aminoantipyrine analogue. More preferably, the coupling agent is 4-aminoantipyrine.
  • “Substantially stable calibration factor” refers to a calibration factor that does not vary by more than about 10%) over a period of about 20 days. Preferably, the calibration factor does not vary by more than about 7% over a period of about 20 days. More preferably, the calibration factor does not vary by more than about 7% over a period of about 40 days. Still more preferably, the calibration factor does not vary by more than 5% over a period of about 40 days. Most preferably, the calibration factor does not vary by more than about 5% over a period of 60 days.
  • Agent that substantially prevents ascorbic acid interference refers to a chemical or biological oxidizing compound. Ascorbic acid interference results from the conversion of ascorbic acid to ascorbate, which acts to reduce a chromogen in a spectrophotometric assay.
  • the chemical or biological agent oxidizes at least 90% of the ascorbate to ascorbic acid in a sample, thereby substantially preventing the interference.
  • the agent oxidizes at least 95% of the ascorbate to ascorbic acid. More preferably, the agent oxidizes at least 99% of the ascorbate to ascorbic acid.
  • Agent that substantially prevents bilirubin interference refers to a chemical or biological oxidizing compound.
  • Bilirubin interference results because bilirubin absorbs light at wavelengths commonly used for spectrophotometric assays.
  • the chemical or biological agent oxidizes at least 90% of the bilirubin in a sample, thereby substantially preventing the interference.
  • the agent oxidizes at least 95% of the bilirubin.
  • the agent oxidizes at least 99% of the bilirubin.
  • the present invention is directed to sets of liquid reagents that provide calibration stability in an enzyme based spectrophotometric assay for the measurement of lactate in patient samples.
  • the reagent sets for the spectrophotometric assay include a lactate oxidase, a peroxidase, a hydrogen donor, an agent that substantially prevents ascorbic acid interference, an agent that substantially prevents bilirubin interference, a coupling agent, a buffer and, optionally, a preservative.
  • lactate oxidase should be specific for L-lactate, soluble, catalase free and highly stable.
  • lactate oxidase that meets those criteria is produced by Streptococcus faecalis. See U.S. Pat. No. 4,166,763.
  • the lactate oxidase used in the present invention is isolated from a microorganism.
  • the lactate oxidase is present in the reagent set at a concentration ranging from about 2.6 U/mL to about 17.5 U/mL.
  • the lactate oxidase is present at a concentration ranging from about 10.0 U/mL to about 17.5 U/mL. More preferably, the lactate oxidase is present at a concentration ranging from about 12.5 U/mL to about 17.5 U/mL. Most preferably, the lactate oxidase is present at a concentration of about 15.0
  • the peroxidase used in the present invention can be isolated from a variety of sources.
  • a nonlimiting list of peroxidase sources includes horseradish, potatoes, figtree sap, turnips (plant peroxidase), milk (lacto peroxidase), white blood corpuscles (verdo peroxidase) and microorganisms. See U.S. Pat. No. 4,166,763.
  • Certain synthetic peroxidases are also suitable for use in the present invention. See Theorell et al. (1950) Ada Chetn. Scand. 4:422-433.
  • the peroxidase is isolated from horseradish.
  • the peroxidase is present in the reagent set at a concentration ranging from about 2.0 U/mL to about 30.0 U/mL.
  • the peroxidase is present at a concentration ranging from about 10 U/mL to about 30.0 U/mL. More preferably, the peroxidase is present at a concentration ranging from about 20 U/mL to about 30.0 U/mL. Most preferably, the peroxidase is present at a concentration of about 24 U/mL.
  • the hydrogen donor used in the present invention is an aniline derivative of the following structure:
  • Ri is hydrogen or alkyl
  • R 2 and R 3 are, independently, hydrogen, alkyl or aryl
  • R is SO 3 M or CO M, wherein M is hydrogen or a cation
  • R 5 is hydrogen or alkyl.
  • Ri is alkyl
  • R and R 3 are hydrogen
  • R 4 is SO 3 M
  • R is alkyl.
  • R ⁇ is methyl, ethyl or propyl
  • R 2 and R 3 are hydrogen
  • R 4 is SO 3 Na
  • R 5 is methyl, ethyl or propyl.
  • R ⁇ is methyl
  • R 2 and R 3 are hydrogen
  • R 4 is SO 3 Na
  • R 5 is methyl (sodium salt of N-ethyl-N-sulfopropyl-m-toluidine).
  • a hydrogen donor used in the present invention will not chemically react with itself or other components in the reagent set to afford a substantial amount of a chromogenic product at about 4 °C.
  • the procedure is repeated. Where the absorbence value at day 21 is more than about 10% different from the absorbence value at day 0, the reagent set does not provide for a relatively stable calibration factor; the hydrogen donor included in the reagent set should not be selected.
  • the hydrogen donor is present in the reagent set at a concentration ranging from about 0.48 g/L (1.75 mM) to about 1.92 g/L (7 mM).
  • the hydrogen donor is present at a concentration ranging from about 0.75 g/L to about 1.5 g/L. More preferably, the hydrogen donor is present at a concentration ranging from about 0.85 g/L to about 1.25 g/L. Most preferably, the hydrogen donor is present at a concentration of about 0.98 g/L.
  • the agent that substantially prevents ascorbic acid interference is either a chemical or biological oxidizing agent. Chemical oxidizing agents that prevent ascorbic acid interference include, without limitation, Fe(III)-HEDTA and iodate. Ascorbate oxidase is an example of a biological oxidizing agent that is used in the present invention.
  • the agent is a biological oxidizing agent. More preferably, it is ascorbate oxidase.
  • the agent that substantially prevents ascorbic acid interference is present in the reagent set at a concentration ranging from about 26.0 U/mL to about 34.0 U/mL.
  • the agent is present at a concentration ranging from about 28.0 U/mL to about
  • the agent is present at a concentration ranging from about 29.0 U/mL to about 31.0 U/mL. Most preferably, the agent is present at a concentration of about 30.0 U/mL.
  • the agent that substantially prevents bilirubin interference is either a chemical or biological oxidizing agent.
  • Bilirubin oxidase is an example of a biological oxidizing agent that is used in the present invention.
  • Chemical oxidizing agents that prevent bilirubin interference include, without limitation, potassium ferrocyanide, sodium ferrocyanide, calcium ferrocyanide, ammonium ferrocyanide and compounds that are capable of generating ferrocyanide.
  • the agent is a chemical oxidizing agent. More preferably, it is potassium ferrocyanide (K [Fe(CN) ]).
  • the agent that substantially prevents bilirubin interference is present in the reagent set at a concentration ranging from about 0.08 g/L to about 0.2 g/L.
  • the agent is present at a concentration ranging from about 0.09 g/L to about 0.15 g/L. More preferably, the agent is present at a concentration ranging from about 0.1 g/L to about 0.14 g/L. Most preferably, the agent is present at a concentration of about 0.13 g/L.
  • the coupling agent is a compound that couples with an oxidized hydrogen donor to provide a chromogen.
  • Examples of coupling agents include, but are not limited to, the following compounds: 4-aminoantipyrine; trisubstituted 4-amino-3-pyrazoline-5-ones, such as l-(2,4,6-trichlorophenyl)-2,3-dimethyl-4-amino-pyrazoline-5-one and l-(3,5- dichlorophenyl)-2,3-dimethyl-4-amino-3-pyrazoline-5-one; and, 4-aminoantipyrine analogues, such as l-phenyl-2,3-dimethyl-4-dimethylamino-3-pyrazoline-5-one.
  • the coupling agent is 4-aminoantipyrine or a 4-aminoantipyrine analogue.
  • the coupling agent is 4-aminoantipyrine.
  • the coupling agent is present in the reagent set at a concentration ranging from about 1.0 mM to about 7.0 mM.
  • the coupling agent is present at a concentration ranging from about 2.0 mM to about 6.0 mM. More preferably, the coupling agent is present at a concentration ranging from about 2.5 mM to about 5.5 mM. Most preferably, the coupling agent is present at a concentration of about 2.9 mM.
  • the buffer is of a suitable buffering capacity to maintain a pH range of about 6.0 to about 7.9 in the reagent set.
  • the buffer system maintains a pH range of about
  • the buffer system maintains a pH range of about 7.5 to about 7.9. More preferably, the buffer system maintains a pH range of about 7.7 to 7.9.
  • Nonlimiting examples of a buffer include phosphate, HEPES, 4-morpholine propanesulfonic acid (MOPS), 2— [tris(hydroxymethyl)methylamino]-l-ethane-sulfonic acid (TES), and TRIS.
  • MOPS 4-morpholine propanesulfonic acid
  • TES 2— [tris(hydroxymethyl)methylamino]-l-ethane-sulfonic acid
  • TRIS TRIS
  • the buffer is a phosphate buffer.
  • the reagent sets of the present invention optionally contain a preservative.
  • preservatives include sodium azide, hydroxybenzoic acid, gentamicin, Thymol and mercury-free preservatives.
  • the preservative is sodium azide.
  • the preservative when it is in the reagent set, is present at a concentration ranging from about 0.05% to about 0.4%.
  • the preservative is present at a concentration ranging from about 0.075% to about 0.3%. More preferably, the preservative is present at a concentration ranging from about 0.085% to about 0.25%.
  • the preservative is present at a concentration ranging from about 0.09% to about 0.2%>.
  • a reagent set of the present invention that does not contain a preservative in the presence of a hydrogen donor, when stored at a temperature of about 35 °C, will provide for a calibration factor that varies by less than about 10% over a period of about 20 days.
  • the reagent set will provide for a calibration factor that varies by less than about 10% over a period of about 40 days. More preferably, the reagent set will provide for a calibration factor that varies by less than about 7% over a period of about 40 days.
  • a reagent set of the present invention that does contain a preservative in the presence of a hydrogen donor, when stored at a temperature of about 4 °C, will provide for a calibration factor that varies by less than about 10%> over a period of about 20 days.
  • the reagent set will provide for a calibration factor that varies by less than about 7% over a period of about 20 days.
  • the reagent set will provide for a calibration factor that varies by less than about 7% over a period of about 40 days.
  • the reagent set will provide for a calibration factor that varies by less than about 5% over a period of about 40 days.
  • the reagent set will provide for a calibration factor that varies by less than 5% over a period of about 60 days.
  • Reagent RI contains NaH 2 PO 4 x H O, Na 2 HPO 4 x 2 H 2 O, sodium azide, ascorbate oxidase and N-ethyl-N-sulfopropyl-m-toluidine, sodium salt as the hydrogen donor.
  • Reagent R2 contains CaCl 2 x 2 H 2 O, NaH 2 PO 4 x H 2 O, Na 2 HPO x 2 H 2 O, sodium azide, K 4 [Fe(CN) 6 ] x 3 H O, 4-AAP, lactate oxidase and peroxidase.
  • Reagent set RI and R2 provides calibration stability in an enzyme based spectrophotometric assay for the measurement of lactate.
  • the absorbence of the reagent set at 660 nm was measured at days 0, 7, 14 and 21. At day 0, the absorbence was 86. At days 7, 14 and 21 the absorbence was, respectively, 83, 83 and 83. Between days 21 and 0, therefore, there was a variation in absorbence of approximately only 3.5%: (86-83)/86.
  • the reagent set consists essentially of reagents R3 and R4.
  • Reagent R3 contains NaH 2 PO 4 x H O, Na 2 HPO 4 x 2 H 2 O, ascorbate oxidase and N-ethyl-N-sulfopropyl-m-toluidine, sodium salt as the hydrogen donor.
  • Reagent R4 contains CaCl 2 x 2 H 2 O, NaH 2 PO 4 x H 2 O, Na 2 HPO 4 x 2 H 2 O, sodium azide, K 4 [Fe(CN) 6 ] x 3 H 2 O, 4-AAP, lactate oxidase and peroxidase.
  • Reagent set R3 and R4 also provides calibration stability in an enzyme based spectrophotometric assay for the measurement of lactate.
  • the absorbence of the reagent set R3 and R4 at 660 nm was measured over a period of 48 days at 35 °C and compared to the absorbence of reagent set RI and R2 under the same conditions.
  • Reagent set RI and R2 exhibited a variation of approximately 23% over a 48 day period; reagent set R3 and R4 is exhibited a variation of approximately 7% over a 48 day period.
  • any method of biological fluid collection and preparation can be used to provide a sample for an L-lactate assay.
  • plasma from blood collected in fluoride-oxalate tubes 2.5 mg sodium fluoride and 2.0 mg potassium oxalate/mL blood
  • Serum should not be used to provide the sample.
  • Cerebrospinal fluid obtained according to standard methods known in the art can be used.
  • lactate levels increase rapidly with physical exercise.
  • the time required for lactate values to return to normal depends on the physical fitness of the subject. A resting period of thirty minutes, however, is sufficient for the typical patient.
  • the blood sample should be drawn from a stasis-free vein. Minimal hemostasis— less than 30 seconds— though, will not affect lactate levels. The person drawing the blood should avoid using a tourniquet if possible.
  • Lactate is stable in separated plasma for 2 days at 2-8 °C or 2 hours at 20-25 °C. Lactate in CSF is stable for 24 hours at 2-8 °C, 3 hours at 20-25 °C or 1 month at -20 °C.
  • LOD lactate oxidase
  • POD peroxidase
  • 4-AAP 4-aminoantipyrine
  • H 2 O 2 hydrogen peroxide
  • TOPS N-ethyl-N-sulfopropyl-m-toluidine
  • TOOS N-ethyl-N- (2-hydroxy-3-sulfopropyl)-m-toluidine
  • NaN 3 sodium azide.
  • Reagent Set R3 and R4 Procedure for Lactate Measurement Using Reagent Set R3 and R4.
  • a biological fluid sample (3 ⁇ L) was dispensed into a 37 °C cuvette (water bath controlled) by a Boehringer Marmheim Hitachi 717 Analyzer.
  • Reagent R3 (Table 1, 250 ⁇ L) was added and mixed with the biological sample.
  • reagent R4 (Table 1, 50 ⁇ L) was added and mixed.
  • the cuvette was allowed to stand for 140-193 seconds. The sample absorbence was measured at 660 nm.

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Abstract

L'invention concerne des ensembles de réactifs liquides présentant un facteur d'étalonnage stable dans un dosage spectrophotométrique à base d'enzymes, ces ensembles permettant d'effectuer une détermination quantitative du lactate dans des échantillons prélevés sur des patients. Ces ensembles de réactifs comprennent une lactate oxydase, une peroxydase, un donneur d'hydrogène, un agent destiné à prévenir sensiblement toute interférence de l'acide ascorbique, un agent destiné à prévenir sensiblement toute interférence de la bilirubine, un agent de couplage, un tampon, et éventuellement un agent de conservation. Cette invention concerne également des méthodes d'utilisation de ces ensembles de réactifs liquides.
PCT/US1999/012174 1998-05-29 1999-05-28 Ensemble de reactifs liquides pour une determination quantitative du l-lactate WO1999061657A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CA002334276A CA2334276C (fr) 1998-05-29 1999-05-28 Ensemble de reactifs liquides pour une determination quantitative du l-lactate
EP99953363A EP1082453B1 (fr) 1998-05-29 1999-05-28 Ensemble de reactifs liquides pour une determination quantitative du l-lactate
DE69940155T DE69940155D1 (de) 1998-05-29 1999-05-28 Flüssiges reagenzienset zur l-lactat-bestimmung

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US8723098P 1998-05-29 1998-05-29
US09/087,230 1998-05-29
US09/094,235 US6030802A (en) 1998-05-29 1998-06-09 Liquid reagent set for L-lactate determination
US09/094,235 1998-06-09

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CN112904011A (zh) * 2019-12-04 2021-06-04 苏州普瑞斯生物科技有限公司 一种血清C1q补体的检测试剂盒及制备方法

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EP1082453B1 (fr) 2008-12-24
EP1082453A1 (fr) 2001-03-14
ES2318905T3 (es) 2009-05-01
US6030802A (en) 2000-02-29
CA2334276C (fr) 2009-12-22
DE69940155D1 (de) 2009-02-05

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